Project Summary There are two critical pacemakers for life: the cardiac pacemaker and the breathing pacemaker, the preBtzinger Complex (preBtC). The preBtC is a cluster of ~3000 neurons in the brainstem that are cyclically active, with each burst of activity initiating a breath. In contrast to the cardiac pacemaker, the molecular and cellular basis of breathing rhythm generation remains unknown, as do diseases associated with it, such as central sleep apnea and sudden infant death. The prevailing model of preBtC rhythm generation, called the `group-pacemaker' model, proposes that each breath is triggered by an emergent preBtC network phenomena. An important assumption of this model is that there are not dedicated breath-initiating neurons. However, based on the observed variety of preBtC neuron firing patterns, including ones that fire just before each breath (pre-inspiratory), and the unexpected molecular and functional diversity of the preBtC neurons I discovered during my Ph.D., I hypothesize that, as in the heart, there are specific neurons that initiate each breath, breathing pacemaker neurons, and propose to identify and characterize them in this research proposal. As a UCSF Sandler Fellow and recipient of the Early Independence Award, I plan to first comprehensively map preBtC cell types with single cell gene expression analysis and identify candidate breathing pacemaker neurons by their expression of the same ion channels important for cardiac pacemaking. Additionally, I plan identify candidate breath-initiating neurons by their anticipated activity during breathing (pre-inspiratory) and their autonomous, rhythmic activity in vitro (pacemaker activity). Lastly, I will identify candidate pacemakers by their proposed connectivity to ~175 preBtC neurons I identified in my Ph.D. that receive breathing pacemaker activity. I predict that these three independent approaches will converge on the same preBtC subtypes, the presumed breathing pacemaker neurons and I will then use intersectional genetic strategies to test if the identified neurons have breathing pacemaker properties: autonomous rhythmic activity, pre-inspiratory activity, ability to initiate a breath, and requirement for breathing. The molecular and functional identification of respiratory pacemaker neurons will be a transformative discovery, leading to the eventual resolution of how respiratory rhythms and arrhythmias, some of the most deadly diseases in infants, are generated. This mechanistic understanding of breathing rhythm generation will provide an avenue to develop pharmacological approaches to control ventilation, which would impact multiple medical fields, especially neonatology and critical care medicine. In my recent Ph.D. work, I have demonstrated extraordinary molecular diversity within the preBtC and demonstrated that small numbers of molecularly distinct preBtC cell types have highly specific functions in the breathing behavior. I am poised to continue this dissection with the objective of identifying the core neurons that initiate a breath and control the pace of breathing.